Cytoplasmic dynein was shown by this laboratory to be responsible for retrograde transport along cytoplasmic microtubules, and has been implicated in retrograde organelle transport as well as poleward chromosome movement. It is a complex of two heavy chains of 410 kD which contain the ATPase active sites, and additional polypeptides of 150, 74, 59, 57, 55, 53, and 40-50 kD whose specific function is unknown. We recently cloned and sequenced cDNAs encoding the entire 150 kD polypeptide and found extensive co-linear homology with the similarly-sized product of the Glued locus in Drosophila. We have also selected a 2.5 kbp cDNA heavy chain clone which was verified by amino acid sequence match and recognizes a 16.5 kb mRNA in brain, testis, and liver. This proposal involves a comprehensive program to clone and sequence cDNAs encoding the several component polypeptides of the rat cytoplasmic dynein complex with the goal of testing the role of cytoplasmic dynein in vivo. The ATP-binding site will be identified and the number of distinct heavy chains in the complex will be determined. The cytoplasmic dynein polypeptides will be over-expressed in cultured mammalian cells and their expression will be inhibited by anti-sense oligonucleotides or expression of antisense RNA in vivo. The effect of altered expression on mitosis will be determined and the specific stage of mitotic arrest will be defined. Organelle movement and organelle redistribution will also be evaluated in these cells using specific markers for endosomes, lysosomes, the ER, mitochondria and the Golgi apparatus. Dominant mutations will be construed in the heavy chain as an alternative approach to interfering with function. It will be determined whether the Clued gene product behaves as a cytoplasmic dynein subunit, and Glued mutants will be evaluated for defects in cell division and organelle transport. Using the mammalian probes generated in this study, cDNAs will be selected encoding other cytoplasmic dynein polypeptides in Drosophila to determine whether they map to regions of known Glued suppressors. These studies are of vital importance in defining the role of cytoplasmic dynein in cell division, and in revealing the extent of its role in the complex interactions of membranous structures within the cell. This knowledge is of fundamental relevance to understanding abnormal cell division as it occurs, for example, in cancer, and the etiology of neurodegenerative diseases.